JP4845363B2 - Orally administered composition and whitening agent - Google Patents

Description

  The present invention relates to an oral administration composition used for foods, cosmetics and the like, and more specifically, an oral administration composition having a whitening effect useful for prevention and improvement of skin pigmentation, stains, freckles, etc. It is related with the whitening agent containing.

  Many people suffer from spots, freckles, skin darkening, and inflammation from a cosmetic point of view. These symptoms are said to be caused by ultraviolet irradiation or oxidative stimulation. When ultraviolet rays are received on the skin surface, active oxygen is generated in the tissue, and when this active oxygen causes cell damage or inflammation, an inflammatory chemical mediator is secreted. A melanin pigment is produced in the melanin-producing granule in the pigment cell mainly by the reaction via the mediator. The melanin pigment produced in this way moves to adjacent keratinocytes and accumulates. It is thought that keratinocytes usually become the stratum corneum due to skin metabolism and eventually fall off the skin. However, it is believed that when melanin pigment production is excessive, it deposits in the epidermis and dermis, resulting in spots and freckles.

  Various methods have been studied in the past for the purpose of preventing spots, freckles, skin blackening and inflammation. As an external preparation, many compounds are used for whitening, and examples thereof include hydroquinone and its derivatives, kojic acid, arbutin, placenta extract, plant extract and the like. On the other hand, ascorbic acids such as ascorbic acid, derivatives thereof and salts thereof have been mainly used as compounds having a whitening effect used for internal use.

  Cysteine is also known to be a compound that prevents pigmentation, and its mechanism is considered as follows. There are two main types of melanin pigments: dark eumelanin and light pheomelanin. Cysteine is involved in the synthesis of pheomelanin, which promotes the synthesis of dark eumelanin promoted by ultraviolet rays. Inhibits and prevents pigmentation.

  However, these compounds alone are not always satisfactory in terms of their effects. In order to solve this problem, (a) tranexamic acid and (b) a whitening composition containing L-cysteine (see, for example, Patent Document 1), (a) coenzyme Q10 and (b) cysteine and / or cystine An oral composition comprising a peptide or protein (for example, see Patent Document 2), or an oral administration composition containing (a) carotenoid and (b) ascorbic acid and / or a salt thereof (for example, , See Patent Document 3) and the like.

On the other hand, β-cryptoxanthin is a naturally occurring pigment, and is a carotenoid that is abundant in Wenzhou oranges. In recent years, various functionalities have attracted attention, and examples thereof include an antioxidant action, an anti-cancer action, an osteoporosis-preventing action, a neurite outgrowth action, and the like. In addition, as described above, β-cryptoxanthin is contained in foods such as Unshu mandarin orange and is a main component of carotenoids present in Japanese blood, and there is no problem in safety even if it is taken orally.
JP 2004-217655 A JP 2004-203812 A JP 2004-26720 A

  Any of the compositions described in Patent Documents 1 to 3 described above is within the range of the whitening effect of the compound alone constituting the composition, and a more satisfactory whitening effect has been demanded.

  An object of the present invention is to provide an oral administration composition and a whitening agent that are safer and have a very excellent whitening effect.

  In view of such circumstances, the present inventors have intensively studied to improve the problems of the prior art, and as a result, found that carotenoid β-cryptoxanthin has an excellent whitening effect, and (a It exhibits excellent whitening effect by combining) cryptoxanthin and (b) cysteine, its derivatives or their salts, and, of course, has a whitening effect that is higher than the additive as well as the action shown by each component alone. The headline and the present invention were completed.

That is, the first of the present invention is (a) cryptoxanthin, and (b) cysteine, N-acetyl-L-cysteine, L-homocysteine, L-cysteic acid, L-homocysteic acid, L-cysteine sulfinic acid, S -Containing sulfino-L-cysteine, cystine (a dimer of cysteine) or a salt thereof, and the mixing ratio of (a) and (b) ((a) :( b)) The gist of the present invention is a skin whitening agent comprising an oral administration composition of 1:10 to 1: 7500 as an active ingredient .

  Since the oral administration composition of the present invention has a high melanin production inhibitory effect, it can exhibit an excellent whitening effect when taken orally, and the whitening agent containing this oral administration composition has an excellent effect. It will be a thing. In particular, it is several times more effective than conventional whitening agents such as conventional cysteine alone, vitamin C, lycopene, and β-carotene.

  The (a) cryptoxanthin in the present invention is a known compound selected from 3-hydroxy-carotene and its stereoisomer, or its fatty acid ester, or its protein conjugate, for example, β-cryptoxanthin, Examples include α-cryptoxanthin and its cis isomer, and cryptoxanthine palmitate having a fatty acid bonded to a hydroxyl group, cryptoxanthine myristate, cryptoxanthin laurate, and the like. Among these, β-cryptoxanthin, α-cryptoxanthin and cis isomers thereof are preferable, and β-cryptoxanthin is most preferable in terms of high whitening effect.

  As a method for obtaining cryptoxanthin, a commercially available product may be used, or extraction and purification from a plant such as Unshu mandarin orange may be used. The method for extracting and purifying cryptoxanthin is not particularly limited, and an example is as follows. That is, after hydrolyzing a precipitate of mandarin orange juice and / or a solvent extract from a powder obtained by dehydrating or drying the precipitate, the hydrolyzate is subjected to β-cryption by high performance liquid chromatography using two types of columns. This is a production method for separating a fraction containing xanthine at 95% by weight or more. Examples of the solvent used for extraction include ketones (acetone, methyl ethyl ketone, diethyl ketone, etc.), alcohols (methanol, ethanol, isopropanol, etc.), and saturated hydrocarbons (n-hexane, n-heptane, etc.). Of these, ketones are preferable, and acetone is most preferable in terms of extraction efficiency.

  In order to hydrolyze the solvent extract, a method of adding an alkali metal hydroxide aqueous solution (potassium hydroxide, sodium hydroxide, lithium hydroxide, etc.) and alcohol (methanol, ethanol) to the extract can be mentioned. .

  As the column, one in which the first column is filled with silica powder having an average particle size of 10 to 80 μm and the second column is filled with octadecylsilane silica having an average particle size of 10 to 80 μm is used. The primary developing solvent introduced into the first column is preferably an organic solvent mainly composed of petroleum ether. The secondary developing solvent introduced into the second column is preferably an organic solvent mainly composed of acetonitrile. (For details, refer to JP 2000-136181 A).

  Cryptoxanthine may be not only a high-purity purified product but also a crude extract. Preferred examples of these crude extracts include enzyme-treated products of citrus fruits and / or squeezed koji or extracts thereof. Examples of citrus fruits are not particularly limited as long as they contain a high content of cryptoxanthin, and specific examples include Wenzhou mandarin oranges, persimmons, mangoes, and papayas. There may be. In addition, amylase, glucoamylase, cellulase, hemicellulase, pectinase, mannanase, xylanase, protease, peptidase, lipase, malation enzyme, and other enzymes were added to these fruits or squeezed koji to increase the cryptoxanthin concentration. A thing may be used. Furthermore, you may use the extract which added the organic solvent with respect to the fruit, the fruit squeezed rice cake, and those enzyme processed products. Examples of the organic solvent used include ketones (acetone, methyl ethyl ketone, diethyl ketone, etc.), alcohols (methanol, ethanol, propanol, isopropanol, etc.), saturated hydrocarbons (n-hexane, n-heptane, pentane, etc.), Halogenated hydrocarbons (such as chloroform), esters (such as methyl acetate and ethyl acetate), aromatic hydrocarbons (such as benzene and toluene), acetonitrile, ethers, pyridines, and polyethers are exemplified.

In addition, as the cysteine derivative (b) in the present invention, N-acetyl-L-cysteine, L-homocysteine, L-cysteic acid, L-homocysteic acid, L-cysteine sulfinic acid, S-sulfino-L- It must be cysteine or cystine (a dimer of cysteine). Furthermore, it is also possible to use peptides and proteins containing a high content of cysteine and cystine. Examples of salts of cysteine and its derivatives include mineral salts such as hydrochloride, nitrate and sulfate, alkali metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, and alkaline earth metal salts. Can do. Of these, L-cysteine is preferred when used as a pharmaceutical, and cystine or cysteine peptide is preferred when used as a food, due to current legal regulations.

  As the method for obtaining (b) cysteine, a derivative thereof or a salt thereof in the present invention, a commercially available product may be used, or the cysteine, the derivative thereof or a salt thereof may be produced based on a known method. As a typical example of the production method, eggshell membranes collected from chicken eggs etc. are dried and powdered, and then acid such as hydrochloric acid, sulfuric acid, nitric acid, acetic acid is added to hydrolyze by acting prostheses such as papain and pancreatin. Can be obtained. Further, whey protein or yeast extract may be used.

  The composition for oral administration of the present invention contains the above-mentioned compound which is an essential component. The compounding amount is preferably 0.01 to 50 mg of cryptoxanthin per day and 1 to 750 mg of cysteine (taken), and 0.1 to 10 mg of cryptoxanthin per day, and More preferably, 10 to 500 mg of cysteine will be administered (taken), 0.5 to 5 mg of cryptoxanthin and 30 to 300 mg of cysteine will be administered (taken) per day Is most preferred.

(A) cryptoxanthin and (b) cysteine, N-acetyl-L-cysteine, L-homocysteine, L-cysteic acid, L-homocysteic acid, L-cysteine sulfinic acid, S- in the composition for oral administration of the present invention The compounding ratio with sulfino-L-cysteine, cystine (a dimer of cysteine) or a salt thereof is (a) :( b) = 1: 10 to 1: 7500 as a mass ratio, and (a): (B) = 1: 10 to 1: 480 is more preferable, and (a) :( b) = 1: 10 to 1: 100 is more preferable.

  In the present invention, the content of each component is a concentration based on a value measured by the following quantitative method. For the content of cryptoxanthin, LC-10A manufactured by Shimadzu Corporation was used as an HPLC apparatus, a Resolve C18 (φ3.9 × 150 mm) column manufactured by Waters Co. was connected, and a sample to which an equal amount of methanol was added was introduced. The mobile phase was analyzed for content at methanol: ethyl acetate = 7: 3, column temperature 30 ° C., flow rate 1.0 ml / min, detection wavelength 450 nm. The content of cysteine was determined by treating the sample with 0.2N sodium citrate buffer (pH 2.2), removing solids by filtration, and then using a strongly acidic cation exchange resin column to perform high performance liquid chromatography (post The fluorescence was detected by the column method) and measured.

  In addition to the above essential components, the composition for oral administration of the present invention may be added with components showing known whitening effects, antioxidants, and active oxygen scavengers. Examples of the whitening agent include arbutin, ascorbic acid and derivatives thereof, and salts thereof. Among these, ascorbic acid is preferable, and the amount to be added is 50 to 3000 mg (taken) per day. What was mix | blended in this way is preferable, and what was mix | blended so that it may administer (taking) 300-2000 mg per day is preferable.

  Antioxidants include ubiquinones such as coenzyme Q10, vitamins A and their derivatives, their salts, vitamins B and their derivatives, their salts, vitamins D and their derivatives, and their salts, vitamins E and derivatives thereof, and salts thereof, and the like. Among these, coenzyme Q10 is preferable, and the amount to be added is such that 5 to 200 mg per day is administered (taken). Preferably, those formulated so as to be administered (taken) 10 to 100 mg per day are preferable.

  Examples of the active oxygen scavenger include carotenoids such as astaxanthin, flavonoids such as catechin, and derivatives thereof. Among these, catechin is preferable, and the amount added is 20 to 1000 mg per day (dose) ) Are preferably blended so as to be administered, and those blended so as to be administered (taken) at 100 to 600 mg per day are preferred.

  The oral administration composition of the present invention can exert a skin whitening effect by taking a mixture of those prepared or obtained as described above orally. At that time, the mixture prepared or obtained above may be used as it is, and excipients used for foods and the like (for example, crystalline cellulose, sucrose fatty acid ester, sucrose, etc.) are added, and the dry granulation tablet method or It can be granulated by a wet granule tableting method or the like, and further tableted to be taken as a tablet.

  In addition, wetting agents, emulsifiers, dispersion aids, surfactants, sweeteners, acidulants, sugar alcohols, flavors, aromatic substances, and other excipients commonly used in liquid foods are added and dissolved to form a liquid state. It can also be used as In addition, capsules, gels, soft capsules, troches, pastes, syrups, etc. may be processed and taken.

  The oral administration composition of the present invention, in addition to taking as it is as described above, includes pharmaceuticals, quasi drugs, general foods, foods for specified health use, health foods, functional foods, etc. Any form may be used as long as it is taken orally, such as food and / or beverage. Such foods and / or beverages are not particularly limited. For example, in addition to the above-mentioned pharmaceutical form, bread, udon, buckwheat, rice and other staple foods, cheese, wiener, sausage, ham , Food products such as seafood products, ice cream, cookies, cakes, jelly, pudding, candy, chewing gum, yogurt, gummy, chocolate, biscuits and other sweets, soft drinks, seasonings, alcoholic beverages, energy drinks, coffee , Tea, milk, fruit juice drinks, soft drinks and the like.

  The composition for oral administration of the present invention can be appropriately determined according to the sex, age, symptoms, administration (dosing) method, number of administration (dosing), administration (dosing) timing, etc. It ’s fine. For example, (a) cryptoxanthin per day is preferably administered 0.01 to 50 mg (take), more preferably 0.1 to 10 mg (take), 0.5 to 5 mg (take) Is particularly preferred. In addition, (b) cysteine, a derivative thereof or a salt thereof is preferably administered (taken) in an amount of 1 to 750 mg per day, more preferably administered (taken) in an amount of 10 to 500 mg (taken). Is more preferable.

  The composition for oral administration of the present invention may be produced as a single preparation containing all the components of the present invention and administered (taken), or may be produced as separate preparations. It is also possible to administer (take) simultaneously or sequentially.

  The second of the present invention is a skin whitening agent comprising the above-mentioned composition for oral administration as an active ingredient. If the content of cryptoxanthin in the whitening agent is 0.000001% by mass or more as a dry solid content, there is no problem in the expression of the effect, preferably 0.000001 to 5% by mass, and more preferably 0.001% by mass. It is 00001-0.05 mass%. The content of cysteine is preferably 0.00001 to 10% by mass, more preferably 0.001 to 5% by mass, and most preferably 0.01 to 1% by mass as a dry solid content.

  Further, as a preferred embodiment of the whitening agent, when (a) cryptoxanthin and (b) cysteine, a derivative thereof, or a salt thereof are combined, a medicinal component that is expected to have a synergistic effect, such as a whitening agent, removal of active oxygen, etc. And whitening agents that contain one or more agents, antioxidants, and anti-inflammatory agents. Specific examples of suitable medicinal components include those shown below.

  Examples of the whitening agent include arbutin, ellagic acid, kojic acid, placenta extract, lucinol, tyramine, vitamin C and derivatives thereof and salts thereof, glutathione and derivatives thereof and salts thereof, lycopene and the like. Among the whitening agents, vitamin C and its derivatives and salts thereof are particularly preferable. Such a whitening agent differs depending on the type, but it may be contained in the whitening agent of the present invention in an amount of about 0.00001 to 10% by mass.

  Examples of the active oxygen scavenger include carotenoids such as astaxanthin and carotene, flavonoids such as catechin, and derivatives thereof. Among the active oxygen scavengers, astaxanthin and carotene are particularly preferable. Although these active oxygen scavengers differ depending on the type, the whitening agent of the present invention may contain about 0.00001 to 5% by mass.

  Antioxidants include ubiquinones such as coenzyme Q10, vitamins A and their derivatives, their salts, vitamins B and their derivatives, their salts, vitamins D and their derivatives, and their salts, vitamins Examples include E and derivatives thereof and salts thereof. Among the above antioxidants, vitamin Es and derivatives thereof and salts thereof are particularly preferable. Although these antioxidants differ depending on the type, the whitening agent of the present invention may be contained in an amount of about 0.00001 to 5% by mass.

Reference example 1
Mice-derived B16 cells (JCRB0202) were inoculated in a 24-well plate together with an appropriate amount of medium at 5 × 10 ⁴ cells / well, and statically cultured at 37 ° C. in a carbon dioxide concentration of 5%. After 1 day, the medium is removed by suction, and cryptoxanthin (EXTRASYNTHESE SA) dissolved in DMSO, L-cysteine (manufactured by Wako Pure Chemical Industries) dissolved in water, or both are adjusted to a predetermined final concentration. The medium was replaced with DMSO was added to the system to which only L-cysteine was added so as to have the same concentration as the system to which cryptoxanthin was added. After culturing for 3 days under the same conditions as described above, the medium was removed, washed 3 times with PBS, and then cells were collected by trypsin treatment. The collected cells were centrifuged and washed with PBS (2000 rpm, 5 minutes), and the medium was removed. Further, the supernatant was removed by centrifugation to obtain a cell pellet. 400 μl of 2N NaOH was added to the pellet and well suspended, followed by sonication for 30 minutes. The crude melanin extract thus obtained was added to a 96-well microplate at 100 μl / well and 3 wells / sample, and the absorbance at 405 nm and 690 nm was measured with a microplate reader. The melanin synthesis inhibition rate was calculated as follows.
Melanin synthesis inhibition rate (%) = {1- (OD405S-OD690S) / (OD405C-OD690C)} × 100
OD405C: Absorbance at 405 nm of crude extract from cells without addition of test substance OD690C: Absorbance at 690 nm of crude extract from cells without addition of test substance OD405S: Absorption of crude extract from cells at addition of test substance Absorbance OD690S at 405 nm: Absorbance at 690 nm of the crude extract from the cells when the test substance was added. Melanoma cells were inoculated into a 96-well plate at 5 × 10 ³ cells / well and cultured under the same conditions as above. . After measuring the absorbance at 450 nm and 690 nm on the third day of the culture, 10 μl of CCK-8 (Dojindo) was added, and the mixture was shaken lightly and incubated at 37 ° C. in a carbon dioxide concentration of 5% for 2 hours. After culturing, the absorbance at 450 nm and 690 nm was measured again, and the cell viability was calculated by the following formula.
Cell viability _{(%) = {(OD 450t} = 2h -OD 450t = 0) - (OD 690t = 2h -OD 690t = 0) / (OD 450t = 2h C-OD 450t = 0 C) - (OD 690t = _2h C-OD _{690t = 0} C)} × 100
OD _{450t = 2h} : Absorbance at 450 nm after addition of CCK at the time of addition of test substance OD _{450t = 0} : Absorbance at 450 nm before addition of CCK at the time of addition of test substance OD _{690t = 2h} : 2 hours after addition of CCK at the time of addition of test substance Absorbance OD at 690 nm after 690 _{t = 0} : Absorbance OD at 690 nm before addition of CCK when test substance is added _{450 h = 2 h} C: Absorbance OD at ₄₅₀ nm after addition of CCK without addition of test substance _{450 t = 0} C: Test Absorbance OD at 450 nm before addition of CCK without addition of substance _{690 t = 2h} C: Absorption OD at 690 nm after 2 hours of addition of CCK without addition of test substance _{906 t = 0} C: 690 nm before addition of CCK without addition of test substance Table 1 shows the above results.

As is clear from the results in Table 1, the melanin synthesis inhibitory effect was higher than the additive effect of each component alone by the simultaneous administration of cryptoxanthin and L-cysteine, and increased dramatically.

Reference example 2
The same experiment as in Reference Example 1 was performed using Wenzhou mandarin orange extract as β-cryptoxanthin. Wenzhou mandarin orange extract was prepared by the following method. 200 g of ethanol was added to 20 g of dried powder of mandarin orange pulp, extracted with stirring for 30 minutes, and filtered through a 0.45 μm PTFE membrane filter. The ethanol in the filtrate was completely evaporated by an evaporator, and the resulting Unshu mandarin orange extract was dissolved in 10 ml of DMSO. Further, sterilization and particle removal were performed with a 0.20 μm PTFE membrane filter to obtain an extract of mandarin orange. Other experimental methods followed Reference Example 1. The results are shown in Table 2.

As is apparent from the results in Table 2, the melanin synthesis inhibitory effect is higher than the additive effect of each component alone by the simultaneous administration of β-cryptoxanthin-containing mandarin orange extract and L-cysteine. Rose to.

Reference example 3
Weiser-Maples male brown guinea pigs 6 weeks old were purchased and pre-bred for 2 weeks, and then the color of the entire back skin was judged and divided into 5 groups per group. Samples were orally ingested daily at the following doses during the study period.

  The back of the guinea pig was depilated with an electric clipper, and two places were created with UV light non-irradiation and irradiation between the midline. For UV irradiation, an FL-20SE lamp (wavelength: 250 to 350 nm, manufactured by Toshiba) was used, and UV-B was irradiated for 9 minutes and 30 seconds. The irradiation time was determined from the weakest irradiation amount (minimum erythema amount) in which erythema was observed 24 hours after the ultraviolet irradiation obtained from the preliminary test.

  The following specimens were dissolved in corn oil and administered daily by gavage.

  In addition, the enzyme-treated Unshu mandarin pulp is made from 8kg of the residue after squeezing the fruit juice from Unshu mandarin (citrus juice lees, moisture content of about 90%). Number: pectinase 5,000u / g, arabanase 90u / g) 10g and cellulase, hemicellulase enzyme cellulase Y-NC (manufactured by Yakult Pharmaceutical Co., Ltd., unit number: cellulase 30,000u / g) The mixture was stirred and allowed to stand at room temperature for 8 hours. This reaction solution is centrifuged, the supernatant is removed, water is added and stirred, the supernatant is removed again by centrifugation, and the drum temperature is set to 110 ° C., rotating at 1 rotation / minute. After drying at a speed, the powder was pulverized by a pulverizer.

Sample 1: No addition (control)
Specimen 2: Enzyme-treated Satsuma mandarin pulp (containing 0.5% β-cryptoxanthin) 8 mg / kg
Sample 3: L-cystine 25 mg / kg (manufactured by Wako Pure Chemical Industries)
Sample 4: (Sample 2) + (Sample 3)
Sample 5: Vitamin C 25 mg / kg (manufactured by Wako Pure Chemical Industries)
Sample 6: Lycopene 1 mg / kg (Wako Pure Chemical Industries)
From the 8th day from the start of the test, the pigment was deposited by irradiating ultraviolet rays once a day, 8 days, 10 days, 13 days, 15 days, 17 days and 19 days in total. . The L ^* value (brightness) of the irradiated part was measured using a color difference meter (CR-300, manufactured by Minolta) before starting irradiation and on the 20th day after starting the test. The above results are shown in Table 3.

As is clear from the results in Table 3, the effect of inhibiting melanin synthesis by the simultaneous administration of cryptoxanthin and cystine was higher than the additive effect of each component alone, and increased dramatically.

Example 1
Tablets were produced in a conventional manner with the following composition (2 tablets as a daily dose).

As for the extract of mandarin orange, 8 kg of the residue after squeezing the fruit juice from mandarin orange (citrus juice lees, water content of about 90%) was freeze-dried, 5 L of ethanol was added thereto, and the mixture was stirred for 1 hour, β-cryptoxanthin Extracted. Filtration was performed to collect the ethanol fraction, and ethanol was distilled off using an evaporator to obtain an extract of mandarin orange. Other components were commercially available.
Wenzhou mandarin orange extract (containing 1 mg of β-cryptoxanthin) 20 mg
L-cysteine 240mg
Crystalline cellulose 80mg
Sucrose fatty acid ester 42mg
Sorbitol 100mg
Titanium oxide 18mg

Example 2
Tablets were produced in the usual manner with the following composition (4 tablets per day).

The enzyme-treated Wenzhou mandarin pulp was manufactured by the same method as Sample 2 in Reference Example 3, and commercially available products were used for the other components.
Enzyme-treated Wenzhou mandarin pulp (containing 1 mg of β-cryptoxanthin) 200 mg
Cystine 120mg
Collagen 30mg
Epigallocatechin gallate 10mg
Crystalline cellulose 100mg
Sucrose fatty acid ester 15mg
Sorbitol 25mg

Claims (1)

(A) cryptoxanthin, and (b) cysteine, N-acetyl-L-cysteine, L-homocysteine, L-cysteic acid, L-homocysteic acid, L-cysteine sulfinic acid, S-sulfino-L-cysteine, cystine (A dimer of cysteine) or a salt thereof, and the mixing ratio of (a) and (b) ((a) :( b)) is from 1:10 to 1: 7500 as a mass ratio. A skin whitening agent comprising an oral composition as an active ingredient.